This project proposes to investigate the molecular mechanisms involved in the growth and differentiation of the intestinal mucosa. The mammalian intestinal mucosa is in a constant state of renewal, characterized by active proliferation of stem cells localized in the crypts, progression of these cells up the crypt-villus axis, and subsequent cessation of proliferation and differentiation into one of the four primary cells types: enterocytes, goblet cells, Paneth cells and enteroendocrine cells. The focus of this grant is an in-depth, molecular analysis of the processes that result in the cessation of cellular proliferation and the induction of cellular differentiation. The investigators have previously shown that: 1) the Ras signaling pathway induces the expression of the terminally differentiated neurotensin (NT/N)gene, 2) differentiation is associated with suppression of Cdk activity, induction of the Cdk inhibitor p21waf1/cip1 and an increase in the retinoblastoma (Rb)-related proteins, and 3) intestinal cell differentiation is associated with a decrease in the expression of Bcl2 proteins. The central hypothesis of this proposal is that intestinal differentiation is regulated by signaling mechanisms involving the Ras pathway, cell cycle-related proteins, and the Bcl2 family of proteins. More specifically, the proposal outlines a detailed and logical approach to address the following questions: 1) to further delineate the role of the Ras signaling pathway in gut differentiation by assessing the specific role of ERK inhibition, Raf overexpression, and the PI3K/Akt pathway, 2) to determine the function of the RB and E2F family of proteins in gut differentiation by analyzing the changes in the Rb proteins, the E2F family of proteins, and the effects of overexpression of Cdk2 and Cdk4, and 3) to determine the contribution of the Bcl2 proteins in intestinal differentiation, as well as the potential role of mitochrondria in intestinal differentiation. This will be important in the understanding of the processes of normal cellular growth and differentiation, which is ultimately important in determining the mechanisms of aberrant cellular growth and differentiation that occur in many disease processes, but particularly those which involve neoplasia.